1. Field of the Invention
The present invention is directed to a laser scanning microscope with an illumination beam path and a detection beam path, where a beamsplitter is provided which reflects the illumination light in direction of the sample and transmits the detection light in direction of the detection arrangement. An additional beamsplitter is provided for reflecting the illumination light and for transmitting the detection light, this additional beamsplitter being arranged in the illumination beam path downstream of the first beamsplitter in the illumination direction. The additional beamsplitter also substantially transmits the illumination light reflected at the first beamsplitter and the detection light, but acquires a wavelength range substantially different from the first beamsplitter with respect to its reflectivity.
2. Description of Related Art
In laser scanning microscopes, laser light of one or more wavelengths λexc,n is deflected onto the sample to excite fluorescence. This excitation light is coupled into the sample by a dichroic mirror (color splitter) which only reflects one or more narrow wavelength bands conforming to the excitation wavelengths but transmits all other wavelengths (DE 19702753A1).
The fluorescent light which is detected and which therefore serves for imaging typically has a broad emission band on the long-wavelength side of the excitation. It is guided in the return direction collinear to the excitation and passes the color splitter with the exception of the narrow wavelength bands which the color splitter is designed to reflect. Excitation light which is reflected back by the sample and which would normally exceed the fluorescence by several orders of magnitude is reflected at the color splitter and is not detected. In this way, this color splitter, designated as the main color splitter (HFT), separates the (strong) excitation light from the (weak) fluorescent light by means of wavelength-selective reflection.
The dichroic color splitters described above are based on the principle of multiple-beam interference at a dielectric multilayer system. In this respect, the angle of incidence of the light has a considerable influence on the reflectance or transmittance. The most common configuration is an incidence of less than 45° relative to the perpendicular, but orientation at an incident angle of less than 10° to the perpendicular (10-degree HFT) is also used and achieves higher suppression ratios of the excitation light.
A plurality of interference filters of this kind are combined on a filter wheel to filter different wavelengths. If different excitation lines are used simultaneously, a multiple bandpass is possible. In so doing, a plurality of narrow reflection bands are realized with a layer system on a filter. For example, it is conventional to combine 488 nm and 633 nm on a filter. The combinations are subject to strict limits because there is a limited quantity of bands that can be realized with a layer system. A compromise must be made between performance (reflectivity/fundamental suppression) and cost. This is the starting point of the invention. The invention is realized through the features of the independent patent claims. Preferred further developments are indicated in the dependent claims.